The interplay of normal and pathological biology that characterizes research on Alzheimer's disease (AD) is well illustrated by the study of a gene product implicated in the fundamental mechanisms of life: presenilin. PS 1 missense mutations cause familial AD, but research during the past 4 years, some of it under this grant, has revealed that PS1 is also required for the genesis of all forms of AD and for the normal processing of diverse receptors in all metazoans. In this first competitive renewal, 7 labs that have each contributed productively to elucidating AD mechanisms are joining forces to apply techniques in molecular and cell biology, genetics, chemistry and animal modeling to answer unresolved questions about the normal and abnormal biology of PS. Our 3 original PIs (Selkoe, Tanzi and Hyman) have joined with 4 talented new investigators (Wolfe, Kovacs, Wasco and Xia) to develop and execute 6 research projects that build on the extensive array of methods and reagents that our labs possess. Our Specific Aims include: 1) to attempt to prove the hypothesis (first developed in this grant) that PS is an intramembrane-cleaving aspartyl protease, by carrying out step-wise addition of the 4 limiting components (PS, nicastrin, aph-1, pen-2) to reconstitute APP and Notch cleavage in mammalian and yeast cells and in pure phospholipid vesicles; 2) to conduct detailed genetic (SNP) association analyses in AD by examining positional candidate genes encoding proteins implicated in PS-related pathways, including PS interactors, gamma-secretase substrates and components, AICD interactors and targets, and PS homologs; 3) to study the interactions of the gamma-secretase components with its substrates by developing novel morphological techniques using advanced fluorescent microscopy methods (FRET and FLIM) that measure protein-protein proximity in living neurons; 4) to characterize the nature of "presenilinase", the unusual activity that converts holoPS into active heterodimers, i.e., how the putative autoproteolysis of PS differs from its gamma-secretase activity and where in the cell "presenilinase" exists; 5) to build upon our recent discovery of a novel gamma-secretase substrate, nectin-1, a synaptic adherens junction protein, to ask whether various PS 1 mutations alter nectin-1 processing, cell-cell aggregation and synapse formation, and whether modulating cell-cell adhesion alters gamma-secretase activity on nectin-1; and 6) to assess the potential signaling function of a key family of PS substrates, APLP1 and APLP2, and see if their intracellular domains differ from APP as regards nuclear translocation, Fe65 binding and patterns of gene regulation. Taken together, these Aims comprise an integrated and potentially far-reaching examination of the biology of the PS/ gamma-secretase complex, its substrates and their role in the mechanisms and treatment of AD.